ex. 26: dna amplification of mycobacterium tuberculosis by pcr is dna replication in a test tube...
TRANSCRIPT
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Ex. 26: DNA Amplification of Mycobacterium tuberculosis by
PCR is DNA replication in a test tube
Objectives ??
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Invented by Kary Mullis, 1983. Nobel Prize 1993.
“I was working for Cetus, making oligonucleotides. They were heady times. Biotechnology was in flower and one spring night while the California buckeyes were also in flower I came across the polymerase chain reaction. I was driving with Jennifer Barnett to a cabin I had been building in northern California. She and I had worked and lived together for two years. She was an inspiration to me during that time as only a woman with brains, in the bloom of her womanhood, can be. That morning she had no idea what had just happened. I had an inkling. It was the first day of the rest of my life.”
- from Karry Mullis’s autobiography at the Nobel e-Museum
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Did He Really Invent PCR?
The basic principle of replicating a piece of DNA using two primers had already been described by Gobind Khorana in 1971:
– Kleppe et al. (1971) J. Mol. Biol. 56, 341-346.
Progress was limited by primer synthesis and polymerase purification issues.
Mullis properly exploited amplification.
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What is the Polymerase Chain Reaction?
• Think of it as a molecular photocopier
• It is a means of selectively amplifying a particular segment of DNA.
• The segment may represent a small part of a large and complex mixture of DNAs: e.g. a specific exon of a human gene.
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A Molecular Photocopier
A photocopier capable of duplicating a part of a sentence:
“The next day was quite a different day. Instead of being hot and sunny, it was cool and misty. Pooh didn’t mind for himself, but when he thought of all the honey the bees wouldn’t be making, a cold misty day always made him feel sorry for them.” A.A. Milne, 1928.
The words in blue must be unique for the copier to locate the correct piece of text.
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How Powerful is PCR?
PCR can amplify a single DNA molecule, e.g. from a single sperm.
PCR can amplify DNA to a usable amount (visible by gel electrophoresis) in ~2 hours.
The template DNA need not be highly purified — a boiled bacterial colony.
The PCR product can be digested with restriction enzymes, sequenced or cloned.
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The Basics of PCR Cycling 30 - 35 cycles, each comprising:
– Denaturation (95°C), 30 sec.
– Annealing (55–60°C), 30 sec.
– Extension (72°C), time depends on product size.
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DolanDNA Learning Center:
PCR Tutorial
DolanDNA Learning Center
on YouTube
Additional narrated PCR Tutorial
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What’s in the Reaction?
1. Template DNA
2. Reaction buffer and magnesium ions
3. Nucleotides (dNTPs)
4. Primers
5. DNA polymerase (usually Taq)
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So Then, it’s Easy?
Cycling performed with three water baths.
Thermal cyclers introduced in 1986.
Early polymerases were not thermostable, so had to be replenished each cycle.
The 37°C temperature caused non-specific priming, resulting in unwanted products.
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Taq (Thermus aquaticus) DNA polymerase first described in 1988.
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Forensic Microbiology• Primer for a specific organism will
allow for detection that particular organism
• Real-time PCR: Newly made DNA tagged with a fluorescent dye; the levels of fluorescence can be measured after every PCR cycle
• Reverse-transcription PCR (RT-PCR): Reverse transcriptase makes DNA from viral RNA or mRNA
RT-PCR with a norovirus primer
Textbook Clinical Focus, p. 266
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Materials needed per 2 tables:Four 200 µl PCR tubes containing PCR beadsMicropipette and tips capable of measuring out 10 µlPatient samples Positive control sampleDistilled water for negative controlEquipment: Nano centrifuge, PCR Thermocycler
Work in a team of 8 students.
Day 1
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Materials needed per table: Amplified PCR products from
last session Bottle of 0.5 X TBE (Tris-
borate/EDTA) buffer 50 ml measuring cylinder,
250ml Erlenmeyer flask Minigel electrophoresis
apparatus with gel tray, spacers, and comb
micropipettes and matching tips Microcentrifuge tube containing
20 µl of 10x gel loading buffer.
Materials shared by whole class:1. Scales, flasks, electrophoresis
grade agarose, cylinder, and TBE buffer at “gel making station”
2. Microwave 3. Power source (4 available per
class)4. Nanofuge (2 per room)5. DNA standard (instructor handles
it)6. “InstaStain” ethidium bromide
staining sheets (handled or supervised by the instructor).
7. Gel documentation system
Part 2 of Day 1Cast, Load, and Electrophorese a 1.5 % Agarose Gel
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Understanding Gel Electrophoresis
Make 30 ml of a 1.5% agarose gel.
How?
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How does gel electrophoresis separate DNA fragments?
• Gel acts as sieve to filter DNA fragments
• DNA fragments are naturally negatively charged (phosphate backbone)
• DNA pulled towards anode (pos. electrode) by electric attraction
• Smaller fragments move faster through the gel and larger fragments move slower
• gel electrophoresis is optimized by adjusting agarose concentration in gel
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- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
+ + + + + + + + + + + + + + + + + + + + + + + + ++ + + + + + + + + +
DNA is negatively charged and therefore repelled from the negative pole and attracted towards the positive pole
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_ _ _ _ _ _ _ _ _ _ _ _
+ + + + + + + + + + + + +
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A Typical Image of an Agarose Gel Under UV Light
Decreasing DNASize
Largest DNA fragments
Smallest DNA fragments
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The Intensity of the Band is Proportional to the Concentration Of DNA
• An important point to remember is that the intensity of the band is proportional to the amount of DNA found in the band
The upper band has far lessDNA when compared to the lowerband. The intensity of the bands are proportional to the amount of DNA at that position in the gel
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Sizing The Fragments of DNA
The sizes of the various fragments can be identified by including a “ladder” in the gel– A ladder is a mixture of DNA fragments of known
size – A ladder is usually run beside the unknown
sample so that the sizes of the various DNA fragments in the sample can be identified
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Marker / Ladder / Size Standard
• Mixture of DNA fragments of selected sizes
• When run in a gel, fragments will separate into distinct bands that can be used as references
• Fragment size always stated as [X] base pairs (bp)
• Two common ladders: 200bp and 1kbp (1000 bp) ladders
• 200 bp ladder composed of a mixture of small fragments (200 to 4000 bp)
• Ladders commercially available
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Sizing a Gel Product
BasePairs(bp)4000300020001600
1000
500
1Kbp Sample
ladder
2000 bp
1000 bp
40003000
20001600
1000
500
Sample 1Kbp
ladder
Size of this Fragment?
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100
200
400
300
600
1200
1500
1000
500
Size In Base Pair (bp)
The size of the fragment is…??
40003000
20001600
1000
500
?
1 kbp ladder was used
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Music video from "Scientists for Better PCR" and Bio-Rad.